The present invention is concerned with novel compounds which are biologically active as inhibitors of the DNA methyltransferase enzyme and consequently useful in the treatment of diseases and conditions which involve unregulated differentiation of cells and cellular processes. The DNA methyltransferase enzyme (EC 2.1.1.37) catalyzes the covalent methylation of the DNA base cytosine at the C5 position of that base. This modification of the base cytosine in a DNA molecule has been shown to play a vital role in the transcriptional inactivation, i.e., silencing of the chromatin as well as in the development and differentiation of cells. The resulting unregulated differentiation of cells and cellular processes is found to be a contributing factor in the development via transformation, and growth of particular cancers and malignancies. The compounds of the present invention hinder the occurrence of the just-described biochemical process and constrain its pathogenic sequelae. As a result, the compounds of Formula (1.0.0) are useful as chemopreventative and chemotherapeutic agents for treating cancers, proliferative diseases such as psoriasis, and hyperplasia.
The DNA methyltransferase enzyme (EC 2.1.1.37), which has been identified as a single gene product of 190 Kd, catalyzes the cofactor S-adenosylmethionine (SAM) dependent methylation of the cytosine base at the C5 carbon of the pyrimidine base. See, e.g., T. H. Bestor and V. M. Ingram, Proc. Nat. Acad. Sci., 80:5559-5563 (1983). This DNA modification has been shown to play a vital role in the transcriptional inactivation, i.e., silencing of the chromatin, as well as in the development and differentiation of cells, as described in more detail in E. Li, T. H. Bestor and R. Jaenisch, Cell, 69:915-926 (1992).
It is important to point out that aberrant changes in DNA methylation patterns as well as DNA methyltransferase activity itself have been implicated in the progression of cancer. There are two proposed mechanisms by which DNA methyltransferase has been correlated with this progression. (A) The first proposed mechanism is the hypermethylation/hypomethylation of key cell cycle regulatory genes and oncogenes including P16, P15, c-myc and P53. See, e.g., M. Schroeder and Mass, M. J., Biochem.and Biophys. Res. Comm., 235:403-406 (1997). (B) The second proposed mechanism is the low frequency DNA methyltransferase mediated deamination of the cytosine base causing a cytosine to thymine point mutation in the DNA sequence. See, e.g., J. -C. Shen, W. M. Rideout III and P. A. Jones, Cell, 71:1073-1080 (1992).
Further work in the art also supports the correlation between DNA methyltransferase activity and the transformation and progression of colon cells to malignant carcinomas in min-/APC (xe2x88x92/xe2x88x92) knock-out mice, as described in more detail in P. W. Laird, L. Jackson-Grusby, A. Fazell, S. L. Dickinson, W. E. Jung,. E. Li, R. A. Wienberg and R. Jaenisch, Cell, 81:197-205(1995).
The present invention relates to heterocyclic bisamidines useful as antiproliferative agents, comprising a compound of Formula (1.0.0): 
and a pharmaceutically acceptable salt thereof, wherein:
x is xe2x80x94C(R14)xe2x80x94; or xe2x80x94Nxe2x80x94;
R1 is independently selected from the group consisting of hydrogen; and (C1-C3)alkyl;
R3, R4, R5, R8, R9, and R10 are each independently selected from the group consisting of hydrogen; and (C1-C3)alkyl;
xe2x80x94orxe2x80x94
R3 and R4 may be taken together, or R8 and R9 may be taken together with the nitrogen atoms to which they are attached, to form an imidazolinyl group; or further together with an additional ring carbon, xe2x80x94CH2xe2x80x94, to form a 1 ,4,5,6-tetrahydropyrimidinyl group;
R14 is independently selected from the group consisting of xe2x80x94H; xe2x80x94NHC(xe2x95x90O)(CH2)mR20; xe2x80x94(CH2)mR20; xe2x80x94CH(CH3)R20; xe2x80x94(CH2)m(C6H3)xe2x80x94R17; xe2x80x94(CH2)m(C6H3)xe2x80x94R20; xe2x80x94(CH2)m(heterocyclyl)xe2x80x94R17; xe2x80x94(CH2)m(heterocyclyl)xe2x80x94R20; xe2x80x94CH2CHxe2x95x90CHR20; xe2x80x94(CH2)mC(xe2x95x90O)NHCHR20R21; and xe2x80x94(CH2)mC(xe2x95x90O)NHxe2x80x94CH2xe2x80x94C(xe2x95x90O)NHCHR20R21;
R15 is independently selected from the group consisting of hydrogen; xe2x80x94OR1; xe2x80x94Oxe2x80x94(C1-C3) alkylenyl-R20; and xe2x80x94OR20;
R17 is independently selected from the group consisting of hydrogen; halogen; (C1-C3)alkyl; xe2x80x94CF3; xe2x80x94CN; xe2x80x94NO2; xe2x80x94N(R1)2; xe2x80x94OH; and (C1-C3) alkyl(C1-C3)alkoxy;
R20 is independently selected from the group consisting of xe2x80x94C(xe2x95x90O)OR1; CH(OH)CH2OH; xe2x80x94C(xe2x95x90O)NH2; and xe2x80x94C(xe2x95x90O)H;
R21 is independently selected from the group consisting of hydrogen; (C1-C6)alkyl; xe2x80x94(CH2)nR22; xe2x80x94CH(CH3)CH2C(xe2x95x90O)OR1; and xe2x80x94CH2xe2x80x94(C6H5);
R22 is independently selected from the group consisting of xe2x80x94H; xe2x80x94NH2; xe2x80x94OR1; xe2x80x94SR1; xe2x80x94CN; xe2x80x94OCH2xe2x80x94(C6H5); xe2x80x94O(CH2)mxe2x80x94OR1; xe2x80x94C(xe2x95x90O)OR1; thienyl; tetrahydropyranyl; xe2x80x94CH(OH)CH2OH; xe2x80x94C(xe2x95x90O)C(CH3)xe2x95x90CH2; xe2x80x94NHC(xe2x95x90O)OCH2xe2x80x94(C6H5); and xe2x80x94S(xe2x95x90O)2R1;
m is an integer independently selected from 1, 2, and 3; and
n is an integer independently selected from 1 through 5, inclusive.
The present invention further relates to a pharmaceutical composition for use as an antiproliferative agent, comprising a therapeutically effective amount of a heterocyclic bisamidine compound of Formula (1.0.0) as above described, together with a pharmaceutically acceptable carrier for said compound. The present invention relates as well to a corresponding method of treating a neoplastic or a non-neoplastic disease characterized by abnormally rapid proliferation of tissue involved in said disease, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula (1.0.0) as above described. Said patient is a mammal, including especially a human. Said neoplastic disease includes but is not limited to melanoma, colon cancer; bladder cancer, non-small cell lung cancer, gliomas, head and neck squamous cell carcinoma, breast cancer, prostate cancer, renal cancer, and nasopharyngeal carcinoma. Said non-neoplastic disease includes but is not limited to psoriasis, Pneumocystis carinii infection, and restenosis.
The present invention still further relates to a pharmaceutical composition for use as a DNA methyltransferase inhibiting agent, comprising a therapeutically effective amount of a heterocyclic bisamidine compound of Formula (1.0.0) as above described, together with a pharmaceutically acceptable carrier for said compound. The present invention relates as well to a corresponding method of treating a neoplastic or a non-neoplastic disease characterized by abnormally rapid proliferation of tissue involved in said disease which is mediated by or associated with abnormally increased levels of DNA methylation, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula (1.0.0) as above described. Said patient is a mammal, including especially a human. Said neoplastic disease includes but is not limited to melanoma, colon cancer; bladder cancer, non-small cell lung cancer, gliomas, head and neck squamous cell carcinoma, breast cancer, prostate cancer, renal cancer, and nasopharyngeal carcinoma. Said non-neoplastic disease includes but is not limited to psoriasis, Pneumocystis carinii infection, and restenosis.
Antineoplastic and antiproliferative agents of the present invention may also be used in the therapy of psoriasis, a non-neoplastic disease of the skin characterized by abnormally rapid proliferation of epidermal cells, as well as for the beneficial treatment of Pneumocystis carinii. Therapeutic agents of the present invention are useful in the treatment of proliferative diseases such as restenosis, in addition to cancer and psoriasis.
The present invention is concerned with novel compositions of matter which are heterocyclic bisamidines useful as antiproliferative agents, comprising a compound of Formula (1.0.0): 
and a pharmaceutically acceptable salt thereof.
The X moiety of the compounds of Formula (1.0.0) has the meaning xe2x80x94C(R14)xe2x80x94 or xe2x80x94Nxe2x80x94. Accordingly, there results an indolyl-2-phenyl moiety with a 3-position substituent R14 as the nucleus of the compounds of Formula (1.0.0) when X has the meaning xe2x80x94C(R14)xe2x80x94; and a benzimidazolinyl-2-phenyl moiety as the nucleus of the compounds of Formula (1.0.0) when X has the meaning xe2x80x94Nxe2x80x94. Where R14 has the meaning of hydrogen, then the resulting nucleus is essentially unsubstituted at the 3-position. These basic nuclei may be represented by partial Formulas (1.1.0) and (1.2.0) as follows: 
Of the two above-depicted nuclei, the indolyl-2-phenyl-3-R14 moiety of Formula (1.1.0) is preferred.
The R1 substituent attached to the 1-position nitrogen atom of either the indolyl-2-phenyl or benzimidazolinyl-2-phenyl nucleus has the meaning of hydrogen or (C1-C3) alkyl, with hydrogen and methyl being the preferred meanings.
As used herein, the term xe2x80x9calkylxe2x80x9d, e.g., xe2x80x9c(C1-C3) alkylxe2x80x9d, is intended to have the meaning of a straight or branched chain alkyl group having the indicated number of carbon atoms. Thus, in the case of xe2x80x9c(C1-C3) alkylxe2x80x9d the intended meaning includes methyl, ethyl, n-propyl, and iso-propyl.
The compounds of Formula (1.0.0) are further characterized by having a carbamimidoyl group attached to both the indolyl or benzimidazolinyl component of the basic nucleus, as well as to the phenyl component of said nucleus. These carbamimidoyl groups may be attached to various carbon atoms of said components. The following configurations are representative of some, but not all of the possible configurations included within the scope of the present invention, as illustrated by partial Formulas (1.3.1) through (1.3.12), inclusive: 
where the symbol xe2x80x9c*xe2x80x9d indicates the points of attachment of the substituents R3, R4, R5, R8, R9, and R10, which are not shown in order to simplify the illustration.
Of the above-depicted configurations, those of partial Formulas (1.3.1) and (1.3.10) are preferred, and that of partial Formula (1.3.1) is most preferred.
The substituents on the carbamimidoyl groups illustrated in partial Formulas (1.3.1) through (1.3.16) above are designated R3, R4, R5, R8, R9, and R10 and in one embodiment they are each independently selected from the group consisting of hydrogen and (C1-C3) alkyl. In this embodiment, accordingly, the carbamimidoyl substituents on the indolyl-2-phenyl and benzimidazolinyl-2-phenyl nuclei are selected from the following group illustrated by partial Formulas (1.4.1) through (1.4.15), inclusive: 
Of the above-described carbamimidoyl moieties, the preferred embodiments are those of partial Formulas (1.4.1), (1.4.2), (1.4.5), (1.4.6), (1.4.9), (1.4.10), and (1.4.13). Of these, the more preferred embodiments are those of partial Formulas (1.4.1), (1.4.5), (1.4.9), and (1.4.10). Of these, the most preferred embodiments are those of partial Formulas (1.4.1), (1.4.5), and (1.4.10).
In another embodiment of the compounds of Formula (1.0.0), R3 and R4 may be taken together, or R8 and R9 may be taken together, with the nitrogen atoms to which they are attached, to form an imidazolinyl group; or further together with an additional ring carbon, xe2x80x94CH2xe2x80x94, to form a 1,4,5,6-tetrahydropyrimidinyl group. These embodiments may be illustrated by the following partial Formulas (1.5.1) through (1.5.4): 
where R5 and Rxe2x80x83have the same meaning as set out further above.
R14 is the substituent which is present on the 3-position carbon atom of the indolyl-2-phenyl nucleus of partial Formula (1.1.0), or on the 3-position nitrogen atom of the benzimidazolinyl-2-phenyl nucleus of partial Formula (1.2.0). R14 is the most important and the most variable in structure of all of the substituents which appear on the compounds of Formula (1.0.0). It is hypothesized that the R14 substituent plays a key role in determining the DNA methyltransferase inhibiting activity of the compounds of Formula (1.0.0). R14 is independently selected from the group consisting of xe2x80x94H; xe2x80x94NHC(xe2x95x90O)(CH2)mR20; xe2x80x94(CH2)mR20; xe2x80x94CH(CH3)R20; xe2x80x94(CH2)m(C6H3)xe2x80x94R17; xe2x80x94(CH2)m(C6H3)xe2x80x94R20; xe2x80x94(CH2)m(heterocycly)xe2x80x94R17; xe2x80x94(CH2)m(heterocyclyl)xe2x80x94R20; xe2x80x94CH2CHxe2x95x90CHR20; xe2x80x94(CH2)mC(xe2x95x90O)NHCHR20R21; and xe2x80x94(CH2)mC(xe2x95x90O)NHxe2x80x94CH2xe2x80x94C(xe2x95x90O)NHCHR20R21.
As used herein, the term xe2x80x9cheterocyclylxe2x80x9d is intended to have the meaning of an aromatic or non-aromatic, mono-cyclic or bicyclic, 3- to 10-membered carbocyclic ring system in which at least one of the carbon atoms of the ring has been replaced by a heteroatom independently selected from N, O and S. Preferably two, and more preferably one heteroatom is present, except that in the case of nitrogen, as many as four N heteroatoms may be present. The heterocyclyl group may comprise one or two fused rings, and further may include a combination of a saturated heterocyclic ring and a non-heterocyclic aryl-fused ring.
In prefered embodiments of the present invention where xe2x80x9cheterocyclylxe2x80x9d refers to a non-aromatic mono-cyclic ring system, its meaning includes, but is not limited to, a member selected from the group consisting of oxiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and benzodioxolane, especially 1,3-benzodioxol-5-yl. In more preferred embodiments of this type, xe2x80x9cheterocyclylxe2x80x9d has the meaning pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl.
The term xe2x80x9cheterocyclylxe2x80x9d also refers to an aromatic mono- or bicyclic ring system comprising a 5- or 6-membered ring containing from 1 to 4 heteroatoms independently selected from N, O, and S, optionally having a 3- or 4-carbon chain attached to adjacent carbons thereof to form a fused 9- or 10-membered aromatic ring system. In preferred embodiments of the present invention where the term xe2x80x9cheterocyclylxe2x80x9d refers to mono- or bicyclic aromatic ring systems, its meaning includes, but is not limited to, furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, oxadiazolyl, thiadiazolyl, parathiazinyl, indolyl, benzo[b]furanyl, benzimidazolyl, benzthiazolyl, quinolinyl, and isoquinolinyl. In more preferred embodiments of this type, xe2x80x9cheterocyclylxe2x80x9d has the meaning of pyrrolyl, imidazolyl, oxazolyl or indolyl.
With regard to all of the above-described meanings of xe2x80x9cheterocyclylxe2x80x9d, in the more preferred embodiments of the present invention, it has the meaning of pyrrolyl, pyrrolidinyl, imidazolyl, oxazolyl, piperidinyl, piperazinyl, morpholinyl, or indolyl.
In the definitions of the R14 moiety, the heterocyclyl group is contained in the definitions: xe2x80x94(CH2)m(heterocyclyi)xe2x80x94R17 and xe2x80x94(CH2)m(heterocyclyl)xe2x80x94R20, where m is an integer independently selected from 1, 2, and 3. Accordingly, it will be appreciated that where a heterocyclyl group is attached to the basic nucleus of a compound of Formula (1.0.0), it will be by means of an alkyleneyl bridge. In preferred embodiments, the alkyleneyl bridge will be methylene.
In addition to the above-mentioned heterocyclyl groups defining the R14 moiety, there is also the meaning of the phenyl groups contained in the definitions: xe2x80x94(CH2)m(C6H3)xe2x80x94R17 and xe2x80x94(CH2)m(C6H3)xe2x80x94R20. As in the case of the heterocyclyl groups, the phenyl groups are also attached to the basic nucleus of a compound of Formula (1.0.0) by means of an alkyleneyl bridge which is preferably methylene.
Both the heterocyclyl and phenyl groups defining the R14 moiety are substituted by a group defined by R17 or by a group defined by R20. The R17 group comprises a member independently selected from the group consisting of hydrogen; halogen; (C1-C3)alkyl; xe2x80x94CF3; xe2x80x94CN; xe2x80x94NO2; xe2x80x94N(R1)2; xe2x80x94OH; and (C1-C3) alkyl(C1-C3) alkoxy. The R17 group also defines the substituents from which a member is selected for any given available position of the benzo-portion of the basic nucleus of a compound of Formula (1.0.0), which comprises a benzo-fused ring system consisting of an indolyl-2-phenyl moiety or a benzimidazolinyl-2-phenyl moiety.
Accordingly, it will be appreciated that the same group of substituents is available for selection with respect to the benzo-portion of the basic nucleus, as it is with respect to a phenyl or heterocycl group attached at the 3-position of said basic nucleus, i.e., the 3-position of the indolyl-2-phenyl moiety or the benzimidazolinyl-2-phenyl moiety. Thus, preferred embodiments of the compounds of Formula (1.0.0) which have incorporated therein different substituent groups of the type just mentioned are illustrated in the following partial Formulas (1.6.1) through (1.6.6), inclusive: 
The R14 substituent group may also have the meaning of xe2x80x94NHC(xe2x95x90O)(CH2)mR20, where m is 1, 2 or 3. Accordingly, R14 includes but is not limited to representative moieties of partial Formulas (1.6.10) through (1.6.13), inclusive: 
Further, the R14 substituent group may also have the meaning of an R20 substituent group connected by way of an alkylene or alkenylene bridge, defined as xe2x80x94(CH2)mR20, where m is 1, 2 or 3; xe2x80x94CH(CH3)R20; or CH2CHxe2x95x90CHR20. Accordingly, R14 includes but is not limited to representative moieties of partial Formulas (1.6.20) through (1.6.29), inclusive: 
The R14 substituent group may further have the meaning of an R20 and an R21 substituent group connected by way of specific moieties defined as (CH2)mC(xe2x95x90O)NHCHR20R21 and xe2x80x94(CH2)mC(xe2x95x90O)NHxe2x80x94CH2xe2x80x94C(xe2x95x90O)NHCHR20R21. Accordingly, R14 includes but is not limited to representative moieties of partial Formulas (1.6.30) through (1.6.33), inclusive: 
The R15 group is attached as a substituent at the 2-position of the phenyl component of the indolyl-2-phenyl or benzimidazolinyl-2-phenyl basic nucleus. The R15 substituent is a member independently selected from the group consisting of hydrogen; xe2x80x94OR1; xe2x80x94Oxe2x80x94(C1-C3)alkylenyl-R20; and xe2x80x94OR20. The R20 substituent, in turn, is a member independently selected from the group consisting of xe2x80x94C(xe2x95x90O)OR1, CH(OH)CH2OH, xe2x80x94C(xe2x95x90O)NH2, and xe2x80x94C(xe2x95x90O)H; where the R21 substituent is a member independently selected from the group consisting of hydrogen, (C1-C6) alkyl, xe2x80x94(CH2)nR22 where n is an integer independently selected from 1 through 5 inclusive, xe2x80x94CH(CH3)CH2C(xe2x95x90O)OR1, and xe2x80x94CH2xe2x80x94(C6H5).
The R22 substituent, which appears in the definition of the R21 substituent group described above, is independently selected from the group consisting of xe2x80x94NH2, xe2x80x94OR1, xe2x80x94SR1, xe2x80x94CN, xe2x80x94OCH2xe2x80x94(C6H5), xe2x80x94O(CH2)mxe2x80x94OR1, xe2x80x94C(xe2x95x90O)OR1, thienyl, tetrahydropyranyl, xe2x80x94CH(OH)CH2OH, xe2x80x94C(xe2x95x90O)C(CH3)xe2x95x90CH2, xe2x80x94NHC(xe2x95x90O)OCH2xe2x80x94(C6H5), and xe2x80x94S(xe2x95x90O)2R1.
Consequently, taking into consideration all of the above-described substituent definitions, the R15 substituent groups include, but are not limited to, the following examples illustrated in partial Formulas (1.7.1) through (1.7.10) inclusive: 
The above description contains an indication of subgeneric areas within the scope of the compounds of Formula (1.0.0) which are preferred. There are also a number of specific embodiments within the scope of the compounds of Formula (1.0.0) which are preferred aspects of the present invention. These specific embodiments include but are not limited to those illustrated in the following Formulas (2.0.0) through (2.0.61) inclusive: 
The above-described compounds of Formula (1.0.0) may be utilized in the form of acids, esters, or other chemical classes of compounds to which the compounds described belong. It is also within the scope of the present invention to utilize those compounds in the form of pharmaceutically acceptable salts derived from various organic and inorganic acids and bases in accordance with procedures well known in the art. Such well-known pharmaceutically acceptable salts include, but are not limited to acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, besylate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecysulfate, ethanesulfonate, fumarate, glucoheptanoate, gluconate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, isethionate, lactate, lactobionate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphonate, picrate, pivalate, propionate, salicylate, sodium phosphate, stearate, succinate, sulfate, sulfosalicylate, tartrate, thiocyanate, thiomalate, tosylate, and undecanoate.
Base salts of the compounds of the present invention include, but are not limited to ammonium salts; alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; salts with organic bases such as dicyclohexylamine, meglumine, N-methyl-D-glucamine, tris-(hydroxymethyl)-methylamine (tromethamine), and salts with amino acids such as arginine, lysine, etc. Compounds of the present invention which comprise basic nitrogen-containing groups may be quaternized with such agents as (C1-C4) alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides, bromides and iodides; di(C1-C4) alkyl sulfate, e.g., dimethyl, diethyl and diamyl sulfates; (C10-C18) alkyl halides, e.g., decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl-(C1-C4) alkyl halides, e.g., benzyl chloride and phenethyl bromide. Such salts permit the preparation of both water-soluble and oil-soluble compounds of the present invention.
Among the above-recited pharmaceutical salts those which are preferred include, but are not limited to acetate, besylate, citrate, fumarate, gluconate, hemisuccinate, hippurate, hydrochloride, hydrobromide, isethionate, mandelate, meglumine, nitrate, oleate, phosphonate, pivalate, sodium phosphate, stearate, sulfate, sulfosalicylate, tartrate, thiomalate, tosylate, and tromethamine.
Multiple salts forms are included within the scope of the present invention where a compound of the present invention contains more than one group capable of forming such pharmaceutically acceptable salts. Examples of typical multiple salt forms include, but are not limited to bitartrate, diacetate, difumarate, dimeglumine, diphosphate, disodium, and trihydrochloride.
The pharmaceutical compositions of the present invention comprise any one or more of the above-described anti-proliferative compounds of the present invention, or a pharmaceutically acceptable salt thereof as also above-described, together with a pharmaceutically acceptable carrier in accordance with the properties and expected performance of such carriers which are well-known in the pertinent art.
The term xe2x80x9ccarrierxe2x80x9d as used herein includes acceptable diluents, excipient, adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include but are not limited to, ion exchange compositions; alumina; aluminum stearate; lecithin; serum proteins, e.g., human serum albumin; phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, e.g., prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal silica; magnesium trisilicate; polyvinyl pyrrolidone; cellulose-based substances; e.g., sodium carboxymethylcellulose; polyethylene glycol; polyacrylates; waxes; polyethylene-polyoxypropylene-block polymers; and wool fat.
More particularly, the diluents, excipient, adjuvants and vehicles used in the pharmaceutical compositions of the present invention comprise members selected from the groups consisting essentially of the following: acidifying and alkalizing agents added to obtain a desired or predetermined pH comprise acidifying agents, e.g., acetic acid, glacial acetic acid, malic acid, and propionic acid, and alkalizing agents, e.g., edetol, potassium carbonate, potassium hydroxide, sodium borate, sodium carbonate, and sodium hydroxide; aerosol propellants required where the pharmaceutical composition is to be delivered as an aerosol under significant pressure, e.g., acceptable halogenated hydrocarbons; nitrogen; or a volatile hydrocarbon such as butane, propane, isobutane or mixtures thereof; antimicrobial agents including antibacterial, antifungal and antiprotozoal agents added where the pharmaceutical composition is topically applied, e.g., antimicrobial agents such as benzyl alcohol, chlorobutanol, phenylethyl alcohol, phenylmercuric acetate, potassium sorbate, and sorbic acid, and antifungal agents such as benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, and sodium benzoate; antimicrobial preservatives added to the pharmaceutical compositions in order to protect them against the growth of potentially harmful microorganisms, e.g., alkyl esters of p-hydroxybenzoic acid, propionate salts, phenoxyethanol, methylparaben sodium, propylparaben sodium, sodium dehydroacetate, benzalkonium chloride, benzethonium chloride, and benzyl alcohol; antioxidants added to protect all of the ingredients of the pharmaceutical composition from damage or degradation by oxidizing agents present in the composition itself or the use environment, e.g., anoxomer, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, potassium metabisulfite, propyl octyl and dodecyl gallate, sodium metabisulfite, sulfur dioxide, and tocopherols; buffering agents used to maintain a desired pH of a composition once established, e.g., calcium acetate, potassium metaphosphate, potassium phosphate monobasic, and tartaric acid; and chelating agents used to help maintain the ionic strength of the pharmaceutical composition and bind to and effectively remove destructive compounds and metals, e.g., edetate dipotassium, edetate disodium, and edetic acid.
Dermatologically active agents are added to the pharmaceutical compositions of the present invention to be applied topically, e.g., wound healing agents such as peptide derivatives, yeast, panthenol, hexylresorcinol, phenol, tetracycline hydrochloride, lamin and kinetin, glucocorticosteroids for treating inflammation, e.g., hydrocortisone, dexamethasone, betamethasone, triamcinolone, fluocinolone and methylprednisolone, retinoids for treating acne, psoriasis, cutaneous aging, and skin cancer, e.g., retinol, tretinoin, isotretinoin, etretinate, acitretin, and arotinoid, immunosuppressive agents for treating inflammation, e.g., dapsone and sulfasalazine; mild antibacterial agents, e.g., resorcinol, salicylic acid, benzoyl peroxide, erythromycin-benzoyl peroxide, erythromycin, clindamycin, and mupirocin, antifungal agents, e.g., griseofulvin, azoles such as miconazole, econazole, itraconazole, fluconazole, and ketoconazole, and allylamines such as naftifine and terfinafine, antiviral agents, e.g., acyclovir, famciclovir, and valacyclovir, antihistamines, e.g., diphenhydramine, terfenadine, astemizole, loratadine, cetirizine, acrivastine, and temelastine, topical anesthetics, e.g., benzocaine, lidocaine, dibucaine, and pramoxine hydrochloride, topical analgesics, e.g., methyl salicylate, camphor, menthol, and resorcinol; topical antiseptics for preventing infection, e.g., benzalkonium chloride and povidone-iodine; vitamins and derivatives thereof such as tocopherol, tocopherol acetate, retinoic acid and retinol.
Further examples of diluents, excipient, adjuvants and vehicles used in the pharmaceutical compositions of the present invention comprise members selected from the groups consisting essentially of the following: dispersing and suspending agents, e.g., poligeenan, povidone, and silicon dioxide; emollients, e.g., hydrocarbon oils and waxes, triglyceride esters, acetylated monoglycerides, methyl and other alkyl esters of C10-C20 fatty acids, C10-C20 fatty acids, C10-C20 fatty alcohols, lanolin and derivatives, polyhydric alcohol esters such as polyethylene glycol (200-600), polyoxyethylene sorbitan fatty acid esters, wax esters, phospholipids, and sterols; emulsifying agents used for preparing oil-in-water emulsions; excipients, e.g., laurocapram and polyethylene glycol monomethyl ether; humectants, e.g., sorbitol, glycerin and hyaluronic acid; ointment bases, e.g., petrolatum, polyethylene glycol, lanolin, and poloxamer; penetration enhancers, e.g., dimethyl isosorbide, diethyl-glycol-monoethylether, 1-dodecylazacycloheptan-2-one, and dimethylsulfoxide (DMSO); preservatives, e.g., benzalkonium chloride, benzethonium chloride, alkyl esters of xcfx81-hydroxybenzoic acid, hydantoin derivatives, cetylpyridinium chloride, propylparaben, quaternary ammonium compounds such as potassium benzoate, and thimerosal; sequestering agents comprising cyclodextrins; solvents, e.g., acetone, alcohol, amylene hydrate, butyl alcohol, corn oil, cottonseed oil, ethyl acetate, glycerin, hexylene glycol, isopropyl alcohol, isostearyl alcohol, methyl alcohol, methylene chloride, mineral oil, peanut oil, phosphoric acid, polyethylene glycol, polyoxypropylene 15 stearyl ether, propylene glycol, propylene glycol diacetate, sesame oil, and purified water; stabilizers, e.g., calcium saccharate and thymol; surfactants, e.g., lapyrium chloride; laureth 4, i.e., xcex1-dodecyl-xcexa9-hydroxy-poly(oxy-1,2-ethanediyl) or polyethylene glycol monododecyl ether.
According to this invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Rh, HCIX or similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a rectal suppository formulation, as described above, or in a suitable enema formulation. Topically active transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate , cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspension in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, hydrofluorocarbons, and/or other conventional solubilizing or dispersing agents.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co-administered.
The dosage and dose rate of a compound of Formula (1.0.0) effective for preventing, inhibiting, suppressing or reducing the unregulated differentiation of cells and cellular processes, and consequent or associated pathogenic processes mediated by the DNA methyl transferase enzyme will depend on a variety of factors, such as the chemical nature and biological activity of the inhibitor, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the observations and conclusions of the treating physician.
For example, where the dosage form is oral, e.g., a tablet or capsule, suitable dosage levels of the compounds of Formula (1.0.0) will be between about 1.0 xcexcg and about 10.0 mg/kg body weight per day, preferably between about 5.0 xcexcg and about 5.0 mg/kg body weight per day, more preferably between about 10.0 xcexcg and about 1.0 mg/kg of body weight per day, and most preferably between about 20.0 xcexcg and about 0.5 mg/kg of body weight per day of the active ingredient.
Where the dosage form is topically administered by means of an ointment, suitable dosage levels of a compound of Formula (1.0.0) will be between about 0.1 xcexcg and about 1.0 mg/kg body weight per day, preferably between about 0.5 xcexcg and about 0.5 mg/kg body weight per day, more preferably between about 1.0 xcexcg and about 0.1 mg/kg of body weight per day, and most preferably between about 2.0 xcexcg and about 0.05 mg/kg of body weight per day of the active ingredient.
Using representative body weights of 10 kg and 100 kg in order to illustrate the range of daily topical dosages which might be used as described above, suitable dosage levels of the compounds of Formula (1.0.0) will be between about 1.0-10.0 xcexcg and 10.0-100.0 mg per day, preferably between about 5.0-50.0 xcexcg and 5.0-50.0 mg per day, more preferably between about 10.0-100.0 xcexcg and 1.0-10.0 mg per day, and most perferably between about 20.0-200.0 xcexcg and about 0.5-5.0 mg per day of the active ingredient comprising a compound of Formula (1.0.0). These ranges of dosage amounts represent total dosage amounts of the active ingredient per day for a given patient. The number of times per day that a dose is administered will depend upon such pharmacological and pharmacokinetic factors as the half-life of the active ingredient, which reflects its rate of catabolism and clearance, as well as the minimal and optimal blood plasma or other body fluid levels of said active ingredient attained in the patient which are required for therapeutic efficacy
Numerous other factors must also be considered in deciding upon the number of doses per day and the amount of active ingredient per dose which will be administered. Not the least important of such other factors is the individual response of the patient being treated. Thus, for example, where the active ingredient is used to treat or prevent psoriasis, and is administered topically via ointment with from one to four applications, will be administered each day, each dose, i.e., ointment application containing from about 50.0 xcexcg to about 10.0 mg of active ingredient.
Included within the scope of the present invention are embodiments comprising compositions which contain, in addition to a compound of the present invention as active ingredient, additional therapeutic agent active ingredients selected from the group consisting essentially of anti-inflammatory corticosteroids; non-steroidal anti-inflammatories; immunosuppressants; immunostimulants; antimetabolites; antipsoriatics, anti-cancer agents, and antidiabetics. Specific compounds within each of these classes may be selected from those listed under the appropriate headings in Comprehensive Medicinal Chemistry, Pergamon Press, Oxford, England, pp. 970-986 (1990); and Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th ed., Hardman, J. G. and Limbird, L. E., eds., McGraw-Hill, 1996, the disclosure of which are incorporated herein by reference in their entireties. Especially preferred active ingredients to be included for use in combination with the compounds of Formula (1.0.0) are anti-inflammatory compounds such as theophylline, sulfasalazine and aminosalicylates; immunosuppressants such as cyclosporin, FK-506, and rapamycin; antimetabolites such as cyclophosphamide and methotrexate; and immunomodulators such as the interferons.
Still further embodiments of the present invention relate to a method of treating a neoplastic or a non-neoplastic disease characterized by abnormally rapid proliferation of tissue involved in said disease, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula (1.0.0) as above described. Said patient is a mammal, including especially a human. Said neoplastic disease includes but is not limited to melanoma, colon cancer; bladder cancer, non-small cell lung cancer, gliomas, head and neck squamous cell carcinoma, breast cancer, prostate cancer, renal cancer, and nasopharyngeal carcinoma. Said non-neoplastic disease includes but is not limited to psoriasis, pneumocyctis caring infection, and restenosis.
The present invention still further relates to a pharmaceutical composition for use as a DNA methyltransferase inhibiting agent, comprising a therapeutically effective amount of a heterocyclic bisamidine compound of Formula (1.0.0) as above described, together with a pharmaceutically acceptable carrier for said compound. The present invention relates as well to a corresponding method of treating a neoplastic or a non-neoplastic disease characterized by abnormally rapid proliferation of tissue involved in said disease which is mediated by or associated with abnormally increased levels of DNA methylation, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula (1.0.0) as above described. Said patient is a mammal, including especially a human. Said patient is a mammal, including especially a human. Said neoplastic disease includes but is not limited to melanoma, colon cancer; bladder cancer, non-small cell lung cancer, gliomas, head and neck squamous cell carcinoma, breast cancer, prostate cancer, renal cancer, and nasopharyngeal carcinoma. Said non-neoplastic disease includes but is not limited to psoriasis, pneumocyctis carinii infection, and restenosis.
Antineoplastic and antiproliferative agents of the present invention may also be used in the therapy of psoriasis, a non-neoplastic disease of the skin characterized by abnormally rapid proliferation of epidermal cells, as well as for the beneficial treatment of Pneumocystis carinii. Therapeutic agents of the present invention are useful in the treatment of proliferative diseases such as restenosis, in addition to cancer and psoriasis.
The above-described methods of treatment of the present invention may employ the compounds of Formula (1.0.0) in the form of monotherapy, but said methods may also be used in the form of multiple therapy in which one or more compounds of Formula (1.0.0) are co-administered in combination with a known anti-inflammatory, immunomodulating, immunostimulating or immunosuppressive agent. The terms xe2x80x9cco-administeredxe2x80x9d or xe2x80x9cco-administrationxe2x80x9d as used herein are intended to mean therapeutic utilization of one or more compounds of Formula (1.0.0) in combination with one or more additional therapeutic agents, including but not limited to, administration of the combination of therapeutic active agents in a single dosage form or in multiple dosage forms representing the same or different routes of administration, said multiple dosage forms being administered at substantially the same time or at different times.
Subsequent to synthesis of any of the above-recited preferred species of the present invention or any other compounds falling within the scope of Formula (1.0.0), the biological activities relating to the DNA methyltransferase inhibitory specificities of said compounds may be determined using one or more of the numerous in vitro and in vivo assays which have been described heretofore in the technical literature pertinent to the art. For example, some of the now very-well established assay methods and models concern measurement of DNA methyltransferase inhibitory activity by determining the concentration of a test candidate inhibitor required to block the binding of DNA methyltransferase contained in a cell lysate to a substrate for said enzyme consisting of poly(dl-dC):(dl-dC). The methyl donor S-adenosyl-L-[methyl 3H] methionine (SAM) is also present. After incubation the methylated poly(dl-dC):(dl-dC) product is collected on a filter such as DEAE-Sephadex, epichlorohydrin crosslinked dextran 2-(diethylamino)ethyl 2-[[2-(diethylamino)ethyl]diethylammonio]ethyl ether chloride hydrochloride, and after drying, the amount of said product bound to said filter is determined by scintillation counting.
The compounds of the present invention may be formulated into pharmaceutical compositions that may be administered orally, parenterally, by inhalation (metered dose inhaler, dry powder inhaler or nebulizer), topically, rectally, nasally, intraocularly, buccally, vaginally or via an implanted reservoir. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
The compounds of Formula (1.0.0) may be prepared in accordance with well-known procedures for carrying out the synthesis of organic compounds. A number of different procedures are available which are fully disclosed in the technical literature and with which the skilled artisan will be familiar. The description which follows of such a synthesis scheme is merely representative and not intended to be in any way a limitation of the scope of the present invention.
In Step a of Synthesis Scheme 1, a 4-formyl benzonitrile having the desired R15 substituent (3.0.1) is reacted with a 4-methyl-3-nitro benzonitrile having the desired R17 substituent (3.0.2), in the presence of a base such as sodium methoxide, in a polar, aprotic solvent such as dimethylsulfoxide (DMSO). The reaction mixture is maintained below room temperature. The adduct which is formed is an alcohol (3.0.3).
In Step b of Synthesis Scheme 1, (3.0.3), which is a secondary alcohol, is treated with Jones reagent, an aqueous solution of chromic acid and sulfuric acid, in a polar, solvent such as acetone. The reaction mixture is maintained below room temperature, and the Jones reagent quickly oxidizes the alcohol (3.0.3) to the corresponding ketone (3.0.4).
In Step c of Synthesis Scheme 1, the desired substituent R14 is attached to ketone (3.0.4), which already includes the desired substituents R15 and R17, by treatment with a bromine reagent which includes the R14 moiety. For example, where it is desired to attach an allyl group as defining R14, the reagent used is allyl bromide. Potassium carbonate, K2CO3, is added to the reaction mixture using a polar, aprotic solvent such as dry acetone, after which the reaction mixture is heated at reflux for from 1 to 3 hours, preferably 2 hours. The product containing the desired R14 substituent, (3.0.5), is isolated for use in the next step of the synthesis.
In Step d of Synthesis Scheme 1, the intermediate (3.0.5) is cyclized to form the indolyl-2-phenyl intermediate (3.0.6). This ring closure is carried out using zinc dust in the presence of acetic acid, with reflux of the reaction mixture.
In Step e of Synthesis Scheme 1, the two nitrile groups attached to the indolyl-2-phenyl intermediate (3.0.6) are converted to the corresponding imino ester (imidate) salts in accordance with the conditions required for carrying out the well-known Pinner Reaction. Intermediate (3.0.6) is dissolved in a dry HCl solution of 2-methoxyethanol to which anhydrous methanol is then added. There results the hydrochloride salt of each imino methyl ester, as shown in (3.0.7).
In Step f of Synthesis Scheme 1, the final product (3.0.8) is obtained as the dihydrochloride salt. Final product (3.0.8) contains the unsubstituted carbamimidoyl/carboxamidine moieties of the compounds of Formula (1.0.0). 
There follows a description of preparation and working examples by means of which certain recited compounds of Formula (1.0.0) have been prepared, and by means of which other compounds falling within the scope of Formula (1.0.0) may be prepared in the future by a person having ordinary skill in this art. Said description is presented for the purpose of aiding the person of ordinary skill in making compounds of the present invention, although the instant specification when used in light of the knowledge and experience of such a person of ordinary skill, should prove to be completely adequate and free of ambiguity. Accordingly, the description which follows is not intended to in any way limit the scope of the present invention, and it should not be used in that way. The claims appended hereto define the intended scope of the present invention.